Fluorescence polarization immunoassay technology is well known in the art as evidenced by the following references:
(1) Dandliker, W. B. and Feigen, G. A., Biochem. Biophys. Res. Commun., 5, 299 (1961); PA0 (2) Jolley, M. E., J. Anal. Toxicol. 5, 236 (1981); PA0 (3) Lakowicz, J. R., "Principles of Fluorescence Spectroscopy", Plenum Press, New York, (1983); PA0 (4) "Immunoassay, a Practical Guide", Chan, D. W. and Perlstein, N. T., Eds. Academic Press, New York, 1987); PA0 (5) Price, C. and Newman, D., Chapter 14 ("Homogeneous Fluoroimmunoassay") in "Principles and Practice of Immunoassay", C. P. Price and D. J. Newman, Eds., MacMillan, New York (1991); and PA0 (6) Wei, A-P. and Herron, J. N., Anal. Chem., 65, 3372 (1993).
Fluorescence polarization immunoassays typically use fluorescently labeled target molecules. These labeled target molecules compete with the target molecules of interest for antibody binding sites. As noted by Wei and Herron, supra, fluorescence polarization immunoassays have not been applied with much success to the detection of large macromolecules such as human chorionic gonadotropin (hcg).
The basis of homogeneous fluoroimmunoassays depends upon the binding of an antibody modulating the emission of a fluorophore coupled to an antigen. A low level of fluorescence polarization resulting from the excitation of a fluorescently labeled antigen is increased as the rotation of the antigen is diminished by binding of antibody. Thus, at low sample antigen concentration, the fluorescence polarization will be greatest, reducing as sample antigen displaces fluorophore from the antibody.
Since fluorescence polarization is a measure of the time-average rotational motion of fluorescent molecules, the fluorescent labeling of antibodies to detect antigen-antibody binding has proved largely ineffective due to the disparity in size between antigen and antibody. Generally, an antibody is so much larger than an antigen that the difference in rotational motion when an antigen binds to the antibody is virtually undetectable. Therefore, conventional fluorescence polarization immunoassay technology has been confined to the fluorescent labeling of antigen or target molecule as opposed to antibody or receptor molecule.